1. Field of the Invention
This invention relates to a novel polypeptide designated kd312 and related polypeptides that have an effect on apoptosis, to novel nucleic acid molecules encoding such polypeptides, and to other related aspects.
2. Description of Related Art
Apoptosis
Normal development and tissue homeostasis in animals require the total cell numbers to be kept in an appropriate range. This is achieved by several highly regulated processes involving cell proliferation, survival, and elimination through programmed cell death (apoptosis). An imbalance between the rates of cell production and cell loss can result in serious human diseases such as cancer, disorders of the immune system, and neurodegenerations (reviewed by Rudin, C. M., and Thompson, C. B., Ann. Rev. Med. 48: 267-81 (1997)).
Apoptosis appears to be an evolutionarily conserved, highly organized program of active cell destruction (reviewed by Miura, M., and Yuan, J., Curr. Topics. Dev. Biol. 32: 139-174 (1996); Vaux, D. L. and Strasser, A., Proc. Natl. Acad. Sci. USA 93: 2239-2244 (1996)). In the nematode Caenorhabditis elegans, 14 genes involved in apoptosis have been identified. Among these, the ced-3 gene encodes a cysteine protease of the capsase family and is a key effector in the cell death pathway. The gene product of ced-4 appears to be an adaptor protein which activates ced-3 upon receiving apoptosis signals (Vaux, D. L., Cell 90: 389-390 (1997). The ced-9 gene, a potent suppresser of programmed cell death, negatively regulates the activity of ced-3, probably through ced-4. In mammalian cells, multiple capsases have been identified and shown to be part of the cell death machinery (Henkart, P. A., Immunity 4: 195-201 (1996)). The bcl-2 proto-oncogene appears to be the prototype of mammalian homologs of ced-9 (Vaux, D. L., Cory, S., and Adams, J. M., Nature 335: 440-442 (1988); Vaux, D. L., Weissman, I. L., and Kim, S. K., Science 258: 1955-1957 (1992); Hengartner, M. O., and Horvitz, H. R., Cell 76: 665-676(1994)).
Other members of the bcl-2 family consist of those (such as bcl-XL) that are functionally similar to bcl-2 which can block apoptosis; and others (bax, for example) that have the opposite activity (Boise, L. H., Gonzalez-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., Mao, X., Nunez, G., and Thompson, C. B., Cell 74: 597-608 (1993); Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J., Cell 74: 609-619 (1993)). Although the molecular mechanism is still unclear, recent evidence showed that bcl-2 can block the release of cytochrome c from mitochondria (Kluck, R. M., Bossy-Weitzel, E., Green, D. R., and Newmeyer, D. D., Science 275: 1132-1136 (1997)). In addition, bcl-2 appears to directly inhibit capsase activation by binding to the mammalian ced-4 homolog (Zou, H., Henzel, W. J., Liu, X., Lutzchg, A., and Wang, X., Cell 90: 405-413 (1997)). Other genes besides the bcl-2 family have also been implicated in programmed cell death as well. For example, the transcription factors c-myc and NF-xcexaB may be involved in transducing signals for cell death or survival (Askew, D. S., Ashmun, R. A., Simmons, B. C., and Cleveland, J. L., Oncogene 6: 1915-1922 (1991); Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L. Z., and Hancock, D. C., Cell 69: 119-128 (1992); Hsu, H., Xiong, J., and Goeddel, D. V., Cell 81: 495-504 (1995); Beg, A. A., and Baldwin, A. S., Science 274: 782-784 (1996); Wang, C-Y., Mayo, M. W., and Baldwin, A. S., Science 274: 784-787 (1996)). The tumor suppresser gene p53, which is mutated in over 50% of human cancers, plays an essential role in radiation induced apoptosis in a wide variety of cell types (reviewed by Carson, D. A., Lancet 346: 1009-1011 (1995)).
Blood Loss and Apoptosis
Massive blood loss may deprive animal organs of most of their oxygen supply and lead to cell damage and both necrotic cell death and apoptosis. It is known that many proteins are synthesized in response to low oxygen tensions (hypoxia). Among these proteins, a few with known functions such as erythropoietin (for stimulating erythroid progenitors), vascular endothelial growth factor (for angiogenesis), or the HAP1 protein (for DNA repair), are all known to assist in cell survival during times of hypoxia. Some of the hypoxia-induced proteins may play important roles in cell survival also through reduction or inhibition of apoptosis. In view of recent evidence indicating that alterations in the apoptosis threshold contribute to the pathological cell death or growth in a number of human diseases such as neurodegenerative disorders, ischemic injury, AIDS, and cancers (Thompson, C. B., Science, 267: 1456-1462 (1995)), it is important to identify key factors that protect cells from apoptotic death.
Although a number of cell death related genes and proteins are now known, there remains a need to identify additional such genes and proteins and to determine their biological activity.
Accordingly, it is an object of the present invention to provide novel compounds that are associated with cell death, especially when caused by hypoxia, in mammals.
It is a further object of the invention to provide a method of treating diseases associated with cell death such as those set forth herein.
These and other objects will be apparent to one of ordinary skill in the art from the present disclosure.
To understand better the molecular events governing apoptosis, screening for genes whose expression level is significantly altered during hypoxia induced by blood loss was carried out.
A gene, kd312, has been isolated from rat kidneys and identified to be a gene highly induced after severe blood loss. This gene was also found to be induced in the liver and thymus of the same animal. It was present in the brain but not detected in the bone marrow, heart, or spleen of this animal. The levels of induction in the kidneys can be correlated to the severity of blood loss. The human homolog of this gene was also isolated. The kd312 protein is well conserved between rats and humans. The deduced amino acid sequence of the rat kd312 protein (280 amino acids) shares 97.5% identity with that of the human counterpart (281 amino acids). The kd312 protein is distantly related to the Ras protein family and the human kd312 is most homologous (33.8%) with the R-Ras member of the human Ras family. Similar to Ras proteins, kd312 carries a C-terminal CAAX motif and a GTP-binding site close to the amino terminus. Unlike the Ras genes, neither the rat nor the human kd312 gene induces focus development following expression in NIH3T3 cells. Expression of kd312 in human embryonal kidney cell line 293 protects the cells from apoptosis similar to the effect observed with expression of the bcl-2 gene in this cell line.
The present invention embodies various aspects, as set forth in the following:
In a first embodiment, the present invention provides a nucleic acid molecule encoding a polypeptide selected from the group consisting of:
(a) the nucleic acid molecule of SEQ ID NO:1 or SEQ ID NO:3;
(b) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:2 or a biologically active fragment thereof;
(c) a nucleic acid molecule that encodes a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID NO:2;
(d) a nucleic acid molecule that hybridizes under stringent conditions to any of (a)-(c) above; and
(e) a nucleic acid molecule that is the complement of any of (a)-(d) above.
In another embodiment, the present invention provides a nucleic acid molecule encoding a polypeptide selected from the group consisting of
(axe2x80x2) the nucleic acid molecule of SEQ ID NO:4 or SEQ ID NO:6;
(bxe2x80x2) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:5 or a biologically active fragment thereof;
(cxe2x80x2) a nucleic acid molecule that encodes a polypeptide that is at least 85 percent identical to the polypeptide of SEQ ID NO:5;
(dxe2x80x2) a nucleic acid molecule that hybridizes under stringent conditions to any of (axe2x80x2)-(cxe2x80x2) above; and
(exe2x80x2) a nucleic acid molecule that is the complement of any of (axe2x80x2)-(dxe2x80x2) above.
In another embodiment, the invention provides vectors comprising these nucleic acid molecules, and host cells, either prokaryotic or eukaryotic, comprising the vectors.
The invention further provides a kd312 polypeptide selected from the group consisting of:
(a) the polypeptide of SEQ ID NO:2;
(b) a polypeptide that is at least 85 percent identical to the polypeptide of (a); and
(c) a biologically active fragment of any of (a)-(b).
The invention further provides a kd312 polypeptide selected from the group consisting of:
(axe2x80x2) the polypeptide of SEQ ID NO:5;
(bxe2x80x2) a polypeptide that is at least 85 percent identical to the polypeptide of (axe2x80x2); and
(cxe2x80x2) a biologically active fragment of any of (axe2x80x2)-(bxe2x80x2).
In another embodiment, the invention provides a process for producing a kd312 polypeptide, wherein the polypeptide may be SEQ ID NO:2 or SEQ ID NO:4 or a biologically active fragment thereof, and wherein the process comprises:
(a) expressing a polypeptide encoded by a kd312 nucleic acid molecule in a suitable host; and
(b) isolating the polypeptide.
The invention further provides anti-kd312 antibodies.
The above and additional related aspects of the invention will be better appreciated by referring to the figures which are described in the following section.